Enzyme Immunoassays for the Determination of Ovine LH and FSH

The development of competitive enzyme immunoassays for ovine plasma LH (oLH) and FSH (oFSH) is described. Standards and plasma samples were preincubated with diluted antiserum to oLH or oFSH and the reacted solution (100 μl per well) was transferred to plates previously coated with oLH or oFSH, respectively. The second antibody used was anti‐rabbit IgG horseradish peroxidase. The measuring range was 0.39–50 ng/ml for each hormone and the 50% relative binding sensitivity was 9 ng/ml for oLH. The respective value for oFSH was 3.5 or 34 ng/ml with different hormone and antibody preparations used for the assay. The enzyme immunoassays were used to determine oLH and oFSH levels in plasma from ewes of two breeds during the oestrous cycle. The assays detected the first FSH surge coincident with the LH surge, the second FSH surge about 24 h later and the periodic fluctuations of FSH concentrations during the luteal phase of the oestrous cycle. These enzyme immunoassays are an efficient and economic alternative to the established radioimmunoassays (RIA) for oLH and oFSH.     

Colour Flow Mapping examination: An useful screening test for the early diagnosis of ductus venosus patency in canine newborns

Ductus venosus (DV) closure plays a key role in hepatic circulation adaptation to postnatal metabolic function, and DV patency might develop a congenital portosystemic shunt (CPSS). The noninvasive Color Flow Mapping (CFM) examination, a validated method to diagnose CPSS in adult dogs, is routinely performed to assess DV closure after birth in humans. This study aimed to describe the feasibility of the ultrasonographic evaluation of the DV after birth and to determine its closure time in healthy Great Dane neonates. Patency of DV in serial Color Flow Mapping (CFM) examinations and bodyweight (BW) were recorded on Days 0‐3‐6‐9 in 24 neonates that were classified as having patent (PDV) or closed ductusvenosus (CDV) basing on CFM signal presence/absence. Since the 3rd day, DV diameter was recorded. Data were analysed by ANOVA (p < 0.05). All dogs resulted healthy 1 year later. The number of PDV and CDV puppies at birth was not different on Day 3 (24 and 0 vs. 22 and 2, PDV and CDV, respectively), whereas it resulted different on Days 6 (24 and 0 vs. 14 and 10) and 9 (24 and 0 vs. 0 and 24); on Day 3, it was different compared to Days 6 and 9; on Day 6, it was different from Day 9. Reduction of DV diameter resulted positively related to neonatal BW growth. The CFM evaluation of DV closure after birth in Great Dane puppies represents a feasible technique. Present results suggest the time of functional closure in normal neonates within 9 days after birth. Thus, CFM examination, as an early screening test for DV patency evaluation, performed 10 days after birth, may identify suspicious dogs at risk that would require further investigations. Further studies are needed to deepen the role of a delayed closure in low bodyweight and preterm puppies.     

The Control of Reactive Oxygen Species Influences Porcine Oocyte In Vitro Maturation

The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H₂O₂ or O₂?^‐ production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O₂?^‐ and H₂O₂ scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O₂?^‐ and H₂O₂ during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.     

Alcohol diluent provides the optimal formulation for calcium chloride non-surgical sterilization in dogs

Background

Surgical castration is widely used to sterilize male dogs, but has significant impacts on time to perform the operation, recovery of the animals as well as cost, which can limit population control programs. Previous research has shown intratesticular injection of calcium chloride dihydrate (CaCl₂) in saline to be a promising alternative to surgery. However, long-term azoospermia was not maintained at dosages low enough to avoid side effects. In the search for an optimized formulation, the current investigation is the first study on long-term sterilization effects of intratesticular injection of CaCl₂ in either lidocaine solution or alcohol in dogs. CaCl₂ at 20% concentration in lidocaine solution or alcohol was administered via intratesticular injection to groups of 21 dogs each. The treated animals were examined at 2, 6, and 12 months for sperm production, blood levels of testosterone, and side effects; at time zero and 12 months for testicular size and semen volume. The experimentally treated animals were compared to a control group receiving saline injection only.

Results

Testicles of dogs treated with CaCl₂ in either diluent significantly decreased in size. After administration of CaCl₂ in lidocaine solution, sterility was achieved for at least 12 months in 75% of treated dogs. However, optimal long-term contraceptive effectiveness was achieved with CaCl₂ in alcohol, which resulted in azoospermia over the 12-month study period. Testosterone levels significantly decreased following treatment with CaCl₂, and sexual activity disappeared. Although testosterone returned to baseline levels by 12 months for the group treated with CaCl₂ in lidocaine, dogs injected with CaCl₂ in alcohol had a 63.6% drop in testosterone level, which remained at the low end of physiological range throughout the study. No adverse effects were noted.

Conclusions

A single, bilateral intratesticular injection of 20% CaCl₂ in 95% ethanol was a reliable method for induction of sterilization in 18–28 kg male dogs in this study. The approach showed long-term efficacy and reduced sexual behavior. This chemical method of sterilization might provide an effective, efficient alternative to surgical castration that can have positive impacts on dog welfare.     

Phosphofructokinase and Malate Dehydrogenase Participate in the In Vitro Maturation of Porcine Oocytes

Oocyte maturation depends on the metabolic activity of cumulus–oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2‐oxoglutarate (5, 10 and 20 mm ) or hydroxymalonate (30, 60 and 100 mm ) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10^?5 and (2.54 ± 0.32) 10^?5, and for MDH, the U were (4.72 ± 0.42) 10^?5 and (4.38 ± 0.25) 10^?5 for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10^?3 and (0.94 ± 0.12) 10^?3, and for MDH (9.08 ± 0.93) 10^?3 and (1.89 ± 0.10) 10^?3 for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2‐oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.     

The resistance of meat chickens vaccinated by aerosol with a live V4 Newcastle disease virus vaccine in the field to challenge with a velogenic Newcastle disease virus

Meat chickens housed on a commercial broiler farm in Australia were vaccinated once at 10 to 11 days-of-age by aerosol with live V4 Newcastle disease virus (NDV) vaccine. Groups of vaccinated and unvaccinated birds were flown to Malaysia, where they were challenged with a virulent strain of NDV. Survival rates in vaccinated chickens challenged 7, 14, 21 or 31 d after vaccination were 0.47, 0.77, 0.97 and 0.92, respectively. All unvaccinated chickens died due to Newcastle disease (ND) following challenge. Chickens in Australia and Malaysia were bled and the serums tested for haemagglutination-inhibiting (HI) antibody to NDV. Many vaccinated birds with no detectable antibody, and all birds with a log2 titre of 2 or greater, survived challenge. The results showed that this V4 vaccine induced protective immunity in a significant proportion of chickens within 7 d of mass aerosol vaccination. This early immunity occurred in the absence of detectable circulating HI antibody. Non-HI antibody mediated immunity continued to provide protection up to 31 d after vaccination. Almost all vaccinated birds were protected within 3 w of vaccination. It is concluded that the V4 vaccine is efficacious and could be useful during an outbreak of virulent ND in Australia.     

The experimental production of big liver and spleen disease in broiler breeder hens

SUMMARY Big liver and spleen disease (BLS) was reproduced experimentally by intravenous (IV) and oral (PO) administration of BLS inocula to susceptible broiler breeder hens 34 to 36 weeks of age. Serological and pathological signs of BLS similar to those seen in the natural disease occurred in inoculated and in-contact birds. Splenomegaly was the earliest and often the only necropsy finding, with hepatomegaly and kidney enlargement occurring in some birds later in the course of the disease. After IV administration, serum antigen was detected between 2 and 4 weeks, and antibody between 3 and 5 weeks. After PO administration, antigen was detected between 2 and 4 weeks, and antibody between 3 and 6 weeks. Antibody persisted in all birds to the end of the experiment (6 weeks), and horizontal transmission probably occurred since in-contact birds developed BLS. Liver probably contained the highest concentration of BLS agent because it had the highest infectivity.     

Osteochondrosis of the elbow: a review of the pathogenesis and a new approach to treatment

SUMMARY To test the hypothesis that joint incongruity contributes to the pathogenesis of elbow osteochondrosis, the left and right radius and ulna of 20 young large breed dogs were measured to determine any variation in length and to observe any incongruity of the elbow joint. Both lame and normal dogs were included in the study. Nine of the 20 dogs had marked disparity in radial and ulnar lengths yet only one had obvious elbow joint incongruity. The use of a sliding osteotomy for the treatment of fragmented coronoid process and a lengthening osteotomy for the treatment of an ununited anconeal process is also discussed. All four dogs treated with a sliding osteotomy showed a marked clinical improvement, and two of the three dogs treated with a lengthening osteotomy showed radiographic fusion of the anconeal process.